RNAs that do not function as messenger RNAs, transfer RNAs or ribosomal RNAs are collectively termed non-coding RNAs (ncRNAs). ncRNAs can range in size from 21-25 nucleotides (nt) up to >10,000 nt, and estimates for the number of ncRNAs per genome range from hundreds to thousands. The functions of ncRNAs, although just beginning to be revealed, appear to vary widely from the purely structural to the purely regulatory, and include effects on transcription, translation, mRNA stability and chromatin structure (G. Storz, Science (2002) 296:1260-1262). Two recent pivotal discoveries have placed ncRNAs in the spotlight: the identification of large numbers of very small ncRNAs of 20-24 nucleotides in length, termed micro RNAs (miRNAs), and the relationship of these miRNAs to intermediates in a eukaryotic RNA silencing mechanism known as RNA interference (RNAi).
RNA silencing refers to a group of sequence-specific, RNA-targeted gene-silencing mechanisms common to animals, plants, and some fungi, wherein RNA is used to target and destroy homologous mRNA, viral RNA, or other RNAs. RNA silencing was first observed in plants, where it was termed posttranscriptional gene silencing (PTGS). A similar phenomenon observed in Fungi was termed quelling. These phenomena were subsequently found to be related to a process in animals called RNA interference (RNAi). In RNAi, experimentally introduced double-stranded RNA (dsRNA) leads to loss of expression of the corresponding cellular gene. A key step in the molecular mechanism of RNAi is the processing of dsRNA by the ribonuclease Dicer into short dsRNAs, called small interfering RNAs (siRNAs), of ˜21-23 nt in length having specific features including 2 nt 3′-overhangs, a 5′-phosphate group and 3′-hydroxyl group. siRNAs are incorporated into a large nucleoprotein complex called an RNA-induced silencing complex (RISC). A distinct ribonuclease component of RISC uses the sequence encoded by the antisense strand of the siRNA as a guide to find and then cleave mRNAs of complementary sequence. The cleaved mRNA is ultimately degraded by cellular exonucleases. Thus, in PTGS, quelling, and RNAi, the silenced gene is transcribed normally into mRNA, but the mRNA is destroyed as quickly as it is made. In plants, it appears that PTGS evolved as a defense strategy against viral pathogens and transposons. While the introduction of long dsRNAs into plants and invertebrates initiates specific gene silencing (Hannon, 2002; Hutvagner, 2002), in mammalian cells, long dsRNA can induce the potent translational inhibitory effects of the interferon response (Samuel, 2001). Short dsRNAs of <30 bp, however, evade the interferon response and are successfully incorporated into RISC to induce RNAi (Zamore et al., Cell, 101(1):25-33 (2000); Elbashir, 2001).
Another group of small ncRNAs, called micro RNAs (miRNAs), are related to the intermediates in RNAi and appear to be conserved from flies to humans (Lau, 2001; Lagos-Quintana, 2001; Rhoades, 2002). To date, all metazoans examined have been found to encode miRNAs. MicroRNAs are initially transcribed as a long, single-stranded miRNA precursor known as a pri-miRNA, which may contain one or several miRNAs, and these transcripts are then processed to ˜70 nt pre-miRNAs having a predicted stem-loop structure. The enzyme Dicer cleaves pre-miRNA to produce ˜20-25 nt miRNAs that function as single-stranded RNAi mediators capable of directing gene silencing (Hutvagner, 2002; McManus, 2002). These small transcripts have been proposed to play a role in development, apparently by suppressing target genes to which they have some degree of complementarity. The canonical miRNAs lin-4 and let-7 influence gene expression by binding to sequences of partial complementarity in the 3′ UTR of mRNA, thereby preventing mRNA translation (McCaffrey, 2002). In recent studies, however, miRNAs bearing perfect complementarity to a target RNA could function analogously to siRNAs, specifically directing degradation of the target sequences (Hutvagner, 2002b; Llave, 2002). Thus, the degree of complementarity between an miRNA and its target may determine whether the miRNA acts as a translational repressor or as a guide to induce mRNA cleavage. The discovery of miRNAs as endogenous small regulatory ncRNAs may represent the tip of an iceberg, as other groups of regulatory ncRNAs likely remain to be discovered.
Numerous recent studies have highlighted the importance of miRNAs in regulating gene expression. miRNAs can “fine-tune” gene expression by binding to nearly perfect complementary sequences in mRNAs, thus preventing their translation. The importance of miRNAs in the regulation of specific genes has been demonstrated in a variety of organisms, where their function impacts such universal cellular pathways as cell death, development, proliferation, and hematopoiesis (Ambros, 2004). Additionally, it has been demonstrated that several animal viruses encode their own miRNAs, which target either cellular or viral mRNAs (Cullen, 2006; Nair, 2006; Sarnow, 2006). Recent studies have further underscored the critical role of miRNAs in the maintenance of cellular homeostasis by demonstrating that miRNAs are misregulated in various forms of cancer. Furthermore, specific tumor types have been found to have specific patterns of miRNA expression, or “miRNA signatures” (Calin, 2006; Calin, 2002; Volinia, 2006; Yanaihara, 2006).
The discovery of particular miRNAs that display altered patterns of expression during other disease conditions would help elucidate the role of specific cellular miRNAs and their corresponding target genes is pathogenesis. Such miRNAs could be used, for example, as therapeutic and diagnostic targets.